TRIP13 AAA-ATPase overexpression in chromosomal instability and breast cancer

描述(申请人提供)：非整倍体和染色体不稳定(CIN)在包括乳腺癌在内的实体肿瘤中非常常见。识别和表征非整倍体癌症中复发的遗传和表观遗传学改变将对癌症的预防、诊断和治疗产生巨大的影响。TRIP13被列为与CIN相关的顶级基因之一(在CIN70签名中的第12位)，在多个微阵列研究中，其在预后不良的乳腺癌中也被观察到过表达。然而，TRIP13的作用机制以及TRIP13过表达在乳腺癌发生发展中的病理意义尚不清楚。我们的初步工作发现，TRIP13是AAA-ATPase的成员之一，是一种新的动粒蛋白，直接与有丝分裂检查点沉默蛋白p31彗星相互作用。在2D培养和软琼脂平板中，TRIP13基因敲除延迟了从中期到后期的转变，并显著降低了乳腺癌细胞的增殖。目前建议的目的是阐明TRIP13的有丝分裂功能，并研究TRIP13过表达与乳腺癌发生的关系。中心假说是，TRIP13过表达促进了不合时宜的有丝分裂检查点沉默，推动了CIN和乳腺上皮细胞癌症的发展。三个具体目标将检验这一中心假设。目的1评估TRIP13在有丝分裂检查点的生化功能。具体地说，我们将测试TRIP13，通过它与p31彗星的相互作用和它的ATPase活性，破坏有丝分裂检查点信号所需的蛋白质复合体。在目标2中，我们将使用可诱导的细胞系来调节TRIP13的表达水平，并确定在2D和3D细胞培养模型中对染色体稳定性和细胞增殖的影响。目的3将在异种移植的小鼠模型中检测TRIP13过表达或敲除对乳腺癌细胞生长的影响。总之，这项拟议的工作将测试TRIP13 ATPase的潜在致癌功能，并评估将TRIP13作为控制乳腺癌的新手段的可能性。这项工作还解决了有丝分裂检查点信号研究中的一些关键知识空白。剖析TRIP13的作用机制是我们理解和利用乳腺癌中非整倍体和CIN的持续努力的重要一步。

DESCRIPTION (provided by applicant): Aneuploidy and chromosomal instability (CIN) are remarkably common in solid tumors including breast cancers. Identifying and characterizing recurring genetic and epigenetic changes in aneuploid cancers will have tremendous impact on cancer prevention, diagnosis and treatment. TRIP13 was ranked as one of the top genes associated with CIN (#12 in the CIN70 signature), and its overexpression was also observed in breast cancers with poor prognosis in multiple microarray studies. However, the working mechanism of TRIP13 and the pathological significance of TRIP13 overexpression in breast cancer development remain unclear. Our preliminary work found that TRIP13, a member of the AAA-ATPases, is a novel kinetochore protein and directly interacts with mitotic checkpoint silencing protein p31comet. TRIP13 knockdown delayed metaphase-to- anaphase transition and significantly reduced breast cancer cell proliferation in 2D culture and soft agar plates. The objective of current proposal is to elucidate mitotic functions of TRIP13 and examine the relationship between TRIP13 overexpression and breast cancer development. The central hypothesis is that TRIP13 overexpression promotes untimely mitotic checkpoint silencing, driving CIN and cancer development in mammary epithelial cells. Three specific aims will test the central hypothesis. Aim 1 will assess biochemical functions of TRIP13 in the mitotic checkpoint. Specifically, we will test that TRIP13, through its interaction with p31comet and its ATPase activity disrupts protein complexes required for mitotic checkpoint signaling. In Aim 2, we will use inducible cell lines to modulate TRIP13 expression level and determine the impact on chromosomal stability and cell proliferation in 2D and 3D cell culture models. Aim 3 will examine the effects of TRIP13 overexpression or knockdown on breast cancer cell growth in xenografted mouse models. Together, the proposed work will test potential oncogenic functions of TRIP13 ATPase and assess the possibility to target TRIP13 as a novel means to control breast cancers. The work also addresses some critical knowledge gaps in mitotic checkpoint signaling studies. Dissecting TRIP13 functioning mechanisms represents an important step in our continuous efforts to understand and exploit aneuploidy and CIN in breast cancers.